vol-1, issue-1 cbnaat a novel diagnostic tool for rapid and specific detection of

INTERNATIONAL JOURNAL OF HEALTH RESEARCH IN MODERN INTEGRATED MEDICAL SCIENCES (IJHRMIMS), ISSN 2394-8612 (P), ISSN 2394-8620 (O), Oct-Dec 2014

Original Article

CBNAAT: a Novel Diagnostic Tool For Rapid And Specific Detection Of

Mycobacterium Tuberculosis In Pulmonary Samples

DS Sowjanya1, Ganeswar Behera2, VV Ramana Reddy3, JV Praveen4

Abstract : Mycobacterium tuberculosis remains to be one of the most significant causes of death from an infectious

agent. Rapid diagnosis of tuberculosis and detection of Rifampicin (RIF) resistance are essential for effective disease

management. CBNAAT (Cartridge Based Nucliec Acid Amplification Test) also known as Gene Xpert MTB/RIF assay

is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical

specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of

rifampin resistance in smear-positive and smear-negative pulmonary specimens obtained from possible tuberculosis

patients. Aim of the study - To assess diagnostic usefulness of Gene Xpert MTB/RIF assay technique in management of

tuberculosis. Material and Methods : This is an Observational study conducted in the department of pulmonary medicine

Maharajahs Institute of Medical Sciences, Vizianagaram between June 2012 and December 2013. Two hundred and

five Sputum samples were obtained from Tuberculosis suspects. All samples were tested on Gene Xpert for MTB/RIF

detection after AFB microscopy. Results : 108(52.68%) sputum samples were AFB smear positive and 96 (47.32%)

were negative. In MTB/RIF assay 144 (70.24%) were MTB positive and 61 (29.76%) were negative. Chi-Square test

was applied; P value is


Gene Xpert test is a semi-quantitative nested real-time PCR

in-vitro diagnostic test with two uses:

(1) The detection of Mycobacterium tuberculosis

complex DNA in sputum samples or concentrated

sediments prepared from induced or expectorated sputum

that are either acid-fast bacilli (AFB) smear positive or

negative.

(2) The detection of Rifampicin resistance associated

mutations of the rpoB gene in samples from patients of

Rifampicin resistance5, 6.

Among the most important diagnostic techniques the Gene

Xpert is one which detects gene mutation (rpoB)

associated with RIF resistance. Hence it is of enormous

importance in the diagnosis of both drug susceptible and

drug resistant cases. Gene Xpert test can be performed on

sputum or bronchial washing samples. Results become

available in less than 2 hours. The rapid detection of

Mycobacterium tuberculosis and its resistance to

Rifampicin (RIFs) allows the physician to make critical

decisions in the management of patient regarding therapy

during the same visit.

The aim of this study is to determine the diagnostic

usefulness of the MTB/RIF assay for the diagnosis of

tuberculosis and rapid detection of rifampicin resistance

in smear-positive and smear-negative pulmonary clinical

specimens.

Materials and Methods : This is an observational study

conducted in the department of Pulmonary medicine,

Maharajahs Institute of Medical Sciences during June

2012-December 2013. CBNAAT, an automated instrument

(figure 1) which works on the principle i.e., sample

processing, nucleic acid amplification, and detection of

the target sequences in simple or complex samples using

real-time PCR and reverse transcriptase PCR.

Figure 1 : CBNAAT instrument

205 Patients who presented with symptoms and signs

suggestive of pulmonary tuberculosis, chest X ray showing

features of pulmonary tuberculosis were included in the

study. Sputum samples from these patients were sent for

AFB staining as well as Xpert MTB/RIF test. Early

morning, deep coughed sputum specimens in sterile

containers were included in the study. Specimens were

stored at 2-8C in freezer till further processing. However,

the specimen can be safely stored at 35C for three days.

After collection, Ziehl-Neelsen (ZN) staining was done

on all samples in department of Microbiology and each

sample was run on Gene Xpert.

Standard Assay Procedure of Gene Xpert: The assay

utilizes single-use plastic cartridges with multiple

chambers that are preloaded with liquid buffers and

lyophilized reagent beads necessary for sample processing,

DNA extraction and heminested rt-PCR.7,8Clinical sputum

samples (or decontaminated sputum pellets) are treated

with sodium hydroxide and isopropanol-containing

sample reagent (SR). The SR is added to the sample

(currently recommended at a 3:1 ratio for sputum pellets

and a 2:1 ratio for unprocessed sputum samples) and

incubated at room temperature for 15 min. The treated

sample is then manually transferred to the cartridge which

is loaded into the Gene Xpert instrument.

Subsequent processing is fully automated. The cartridge

incorporates a syringe drive, a rotary drive and a filter

upon which M. tuberculosis bacilli are deposited after

being liberated from the clinical material. The test platform

employs a sonic horn that inserts into the cartridge base

to cause ultrasonic lysis of the bacilli and release of the

genetic material. The assay then amplifies a 192 bp

segment of the rpoB gene using a hemi-nested rt-PCR

reaction8,9. Mycobacterium tuberculosis is detected by the

five overlapping molecular probes (probes AE) that

collectively are complementary to the entire 81 bp rpoB

core region8, 9.

M. tuberculosis is identified when at least two of the five

probes give positive signals with a cycle threshold (CT)

of d38 cycles and that differ by no more than a

prespecified number of cycles. The basis for detection of

rifampicin resistance is the difference between the first

(early CT) and the last (late CT) M. tuberculosis-specific

beacon (CT). The system was originally configured such

that resistance was reported when CT was >3.5 cycles

and sensitive if d3.5 cycles.

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Results

ZN staining was done for 205 samples of the patients who

were having history suggestive of pulmonary tuberculosis.

Out of these 108(52.68%) sputum samples were AFB

smear positive and 96(47.32%) were negative. (Table 1)

Then all the samples were tested on Gene Xpert MTB/

RIF assay. Out of the 205 sputum samples of the patients

having history suggestive of pulmonary tuberculosis, 144

(70.24%) were MTB positive and 61 (29.76%) were

negative. The MTB/RIF test detected the agent in 108 out

of 109 sputum smear positive cases, and 36 out of 96

sputum smear negative cases.

Table 1.

MTB +ve 108 36 144

MTB - ve 1 60 61

Total 109 96 205

samples

Gene Xpert Sputum forAFB+Ve

Sputum forAFB-Ve

Totalsamples

The results of Gene Xpert and ZN staining are compared

in our study. It is evident from the table that Gene Xpert

MTB/RIF is more useful than ZN staining. As compared

to ZN staining it can detect MTB even in 1ml of sputum.

The second important advantage of Gene Xpert is that it

also detects Rifampicin (RIF) resistance and helps us to

diagnose multi-drug resistance tuberculosis (MDR TB).

In table2. 4 patients were rifampicin resistant out of 205

(1.9%) suspected cases, which was confirmed with drug

susceptibility.

Table 2.

NOT 105 96 201DETECTED

DETECT 4 0 4ED

Totalsamples 109 96

RIFResistance

MTB+Ve MTB-VeTotal

samples

Discussion

In this study, the performance of the MTB/RIF assay with

pulmonary specimens obtained during the clinical routine

was compared with AFB staining. In our study, the MTB/

RIF test detected the agent in 144 out of 205 pulmonary

specimens (70.24 % detection rate) whereas sputum for

AFB was able to detect only 108 out of 205 pulmonary

specimens (52.68% detection rate). Out of 109 sputum

smear positive cases, tuberculosis was detected in 108

cases, by MTB/RIF test and out of 96 sputum smear

negative cases 36 cases were detected.

A previous study found that the MTB/RIF assay had a

calculated limit of detection of 131colony-forming

units(CFU)/ml of sputum and was able to detect as few as

10 CFU/ml of sputum in 35% of samples compared with

approximately 10,000 CFU/ml with conventional smear

microscopy10.

The MTB/RIF test is easy to perform and is less dependent

on the users skills. Routine staff with minimal training

can use the test. Technicians can be trained in 1-2 days;

Only 2 steps (addition of buffer and sputum sample) are

manual and the rest of the steps are automated,The results

are available within 90 minutes. Each table top-sized

module can process 4 samples daily (larger modules can

run 200 tests in an 8-hour day), and because it is a closed

system, biosafety and contamination concerns are

minimized. It has a short turn-around time and

simultaneously detects M. tuberculosis and RIF resistance

in less than 2 hours. Although the MTB/RIF test could be

a useful tool for rapid identification of RIF-resistant M.

tuberculosis, especially in smear-positive clinical samples,

the test results must always be confirmed by culture and

DST.

Conclusion

We concluded that as compared to sputum AFB

microscopy, Gene Xpert is more sensitive and specific not

only for acid fast bacilli (AFB) detection but also for

rifampicin (RIF) resistance. Routine staff with minimal

training can use this system. It also helps to avoid

injudicious use of anti-tuberculosis drugs.Statistical Analysis : All results were analyzed statistically

by applying chi-square test. P value was


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Financial Support : Declared None

Conflict of Interest : Declared None

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